The present invention relates to a polypeptide having β-glucosidase enzymatic activity, to a polynucleotide encoding the polypeptide, to nucleic acid constructs carrying the polynucleotide, to transformed or infected cells, such as yeast cells, and organisms expressing the polynucleotide and to various uses of the polypeptide, the polynucleotide, cells and/or organisms, including, but not limited to, producing a recombinant polypeptide having β-glucosidase enzymatic activity, increasing the level of aroma compounds in alcoholic beverages, as well as other fermentation products of plant material, hydrolyzing cellobiose and thus increasing the level of fermentable glucose, to increase production of alcohol, such as ethanol from plant material, increasing the aroma released from a plant or a plant product, and hydrolysis or transglycosylation of glycosides.
Abbreviations used herein include: BGL1—Aspergillus niger B1 β-glucosidase; bgl1—a cDNA encoding same; 2FGlcF—2-deoxy-2-fluoro β-glucosyl fluoride; DNP—2,4-dinitrophenol; DNPGlc—2,4-dinitrophenyl β-D-glucopyranoside; pNP—p-nitrophenol; pNPGlc—p-nitrophenyl β-D-glucopyranoside; MUGlc—4-methylumbeliferyl-β-D-glucopyranoside; YNB—yeast nitrogen base without amino acids; and X-glu—5-bromo-4-chloro-3-indolyl β-D-glucopyranoside.
β-Glucosidases (EC 3.2.1.21; β-D-glucoside glucohydrolase) play a number of different important roles in biology, including the degradation of cellulosic biomass by fungi and bacteria, degradation of glycolipids in mammalian lysosomes and the cleavage of glucosylated flavonoids in plants. These enzymes are therefore of considerable industrial interest, not only as constituents of cellulose-degrading systems, but also in the food industry (2, 3).
Aspergillus species are known as a useful source of β-glucosidases (4-6), and Aspergillus niger is by far the most efficient producer of β-glucosidase among the microorganisms investigated (4). Shoseyov et al. (7) have previously described a β-glucosidase from Aspergillus niger B1 (CMI CC 324626) which is active at low pHs, as well as in the presence of high ethanol concentrations. This enzyme effectively hydrolyzes flavor-compound glycosides in certain low-pH products, such as wine and passion fruit juice, thereby enhancing their flavor (8-12), and is particularly attractive for use in the food industry, as A. niger is considered non-toxic (3). In addition, β-glucosidase was found useful in enzymatic synthesis of glycosides (13-15). Other A. niger β-glucosidases have also been purified (16-18), however, differences in their properties have been reported, including ranges of molecular weights (116-137 kDa), isoelectric points (pI values of 3.8-4) and pH optima (3.4-4.5). Indeed, at least two β-glucosidases, with distinct substrate specificities, have been identified in commercial A. niger β-glucosidase preparations (19). Attempts to clear this confusion by cloning and expression of a functional A. niger β-glucosidase gene in S. cerevisiae has been previously reported (20), however the protein was not characterized, and the sequence was not published.
Glycosidases have been assigned to families on the basis of sequence similarities, there now being some 77 different such families defined containing over 2,000 different enzymes (21, see also the CAZy (Carbohydrate Active EnZymes) website, at the Architecture of Fonction de Macromolecules Biologiques of the Centre National de la Recherche Scientifique website. With the exception of the glucosylceramidases (Family 30), all simple β-glucosidases belong to either Family 1 or 3. Family 1 contains enzymes from bacteria, plants and mammals, including also 6-phospho-glucosidases and thioglucosidases. Furthermore, most Family 1 enzymes also have significant galactosidase activity. Family 3 contains β-glucosidases and hexosaminidases of fungal, bacterial and plant origin. Enzymes of both families hydrolyze their substrates with net retention of anomeric configuration, presumably via a two-step, double-displacement mechanism, involving two key active site carboxylic acid residues (for reviews of mechanism, see 22-24). In the first step, one of the carboxylic acids (the nucleophile) attacks at the substrate anomeric center, while the other (the acid/base catalyst) protonates the glycosidic oxygen, thereby assisting the departure of the aglycone. This results in the formation of a covalent α-glycosyl-enzyme intermediate. In a second step this intermediate is hydrolyzed by general base-catalyzed attack of water at the anomeric center of the glycosyl-enzyme, to release the β-glucose product and regenerate free enzyme. Both the formation and the hydrolysis of this intermediate proceed via transition states with substantial oxocarbenium ion character.
Given that Family 3 contains fungal enzymes of similar mass, including those from other Aspergillus sp., it is likely that the Aspergillus niger β-glucosidase would be a member of this family. Mechanistic information on this family is relatively sparse: the best characterized being the glycosylated 170 kDa β-glucosidase from Aspergillus wentii. By labeling the active site with conduritol B-epoxide, this enzyme was shown to carry out hydrolysis, with net retention of anomeric configuration. This study has demonstrated that the labeled aspartic acid residue was the same as that derivatized by the slow substrate D-glucal (1, 25). Furthermore, it was shown that the 2-deoxyglucosyl-enzyme, trapped by use of D-glucal, was kinetically identical to that formed during the hydrolysis of PNP-2-deoxy-β-D-glucopyranoside (26). Further detailed kinetic analysis of the enzyme was performed by Legler et al. (27), including measurement of Hammett relationships, kinetic isotope effects and studies of the binding of potent reversible inhibitors, such as gluconolactone and nojirimycin.
While reducing the present invention to practice, the β-glucosidase protein was isolated from Aspergillus niger, purified, cloned, sequenced, expressed in yeast host cells and its enzymatic function characterized. In addition, the protein as well as signal peptide fused thereto and optionally an endoplasmic reticulum retaining peptide fused thereto were expressed in transgenic plants and the release of aroma substances therefrom following homogenization monitored. The enzyme encoded by the isolated gene, as described above, is of known usefulness in plant and/or plant products, as well as in biotechnological processes, including the food industry. Several unexpected advantages were uncovered, including, but not limited to, pH and temperature stability of the β-glucosidase from Aspergillus niger, requirement for a signal peptide for obtaining catalytic activity when expressed in plants. Advantage for an endoplasmic retaining peptide or for a lack thereof when expressed in plants, depending on the application.